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Research Article | Open Access
Secondary Metabolite Profile, Antioxidant Capacity, and Mosquito Repellent Activity of Bixa orellana from Brazilian Amazon Region
The Brazilian flora was widely used as source of food and natural remedies to treat various diseases. Bixa orellana L. (Bixaceae), also known as annatto, urucù, or achiote, is a symbol for the Amazonian tribes that traditionally use its seeds as coloured ink to paint their bodies for religious ceremonies. The aim of this study was to investigate the volatile organic compounds (VOCs) profile of B. orellana fresh fruits (in vivo sampled), dried seeds, wood, bark, and leaves analyzed with Headspace solid-phase microextraction coupled with gas chromatography and mass spectrometry. A screening on phenolic content (the Folin-Ciocalteu assay) and antiradical activity (DPPH assay) of seeds was also conducted. In addition, the repellent properties of seed extracts against Aedes aegypti L. were investigated. Volatile compounds detected in B. orellana samples consisted mainly of sesquiterpenes, monoterpenes, and arenes: α-humulene is the major volatile compound present in seed extracts followed by D-germacrene, γ-elemene, and caryophyllene. B. orellana proved to be a good source of antioxidants. Preliminary data on repellency against A. aegypti of three different dried seed extracts (hexane, ethanol, and ethanol/water) indicated a significant skin protection activity. A protection of 90% and 73% for hexane and ethanol/water extracts was recorded.
The Brazilian Amazon regions represent one of the world’s richest pockets of biodiversity. Rich in endemic plants, tropical forests are known to host thousands of edible, medicinal and aromatic plant species . These regions are also populated by numerous Indio tribes renowned for their traditional knowledge in using herbs as a source of food and natural remedies for different illnesses . In 2006, Brazil’s Ministry of Health established a policy regulating the use of medicinal plants and phytotherapeutic resources (Política Nacional de Plantas Medicinais e Fitoterápicos), aimed at improving healthcare and sustainable uses of biodiversity .
B. orellana L. (Bixaceae), a shrub native to Central and South America, also known as annatto, urucù, or achiote, is a symbol for the Amazonian tribes that traditionally use its seeds as coloured ink to paint their bodies for religious ceremonies . Moreover, it is believed that the original Aztec chocolate beverage contained annatto seeds in addition to cocoa . The term “annatto” in industrialized countries is commonly referred to B. orellana seed extract containing carotenoid-type pigments, widely used to dye an assortment of foods, textiles, and body care products . Recently, B. orellana extracts have also been shown to be a viable option for commercial exploitation as a replacement for synthetic dyes and pigments in dyeing and finishing of leather . Two carotenoids give annatto its unique red colour: bixin and norbixin. Bixin (methyl (9-cis)-hydrogen-6,6-diapo-W,W-carotenedioate) is the main responsible for the orange-red colour of the seeds, and extracts of annatto represent approximately 80% of its total carotenoids [5, 7]. Therefore, B. orellana is a well-known commercial crop. The main commercial producers are tropical countries including Jamaica, Mexico, and the Philippines, with Peru, Brazil, and Kenya as the main sources of supply . About 80% of the world production, which ranges between 10,000 and 11,000 tons of seeds per year, is utilized by the USA and Western Europe where it is further processed and used to impart the characteristic orange-yellow colour to butter, margarine, and cheese .
It is a matter of fact that almost all the organs of B. orellana are used in traditional medicine to obtain remedies to treat various diseases . In Brazil, B. orellana leaves, roots, and seed extracts are popular as aphrodisiac medicines as well as a remedy to treat fevers, inflammatory conditions, and parasitic diseases. The entire plant is used against dysentery. Extracts of leaves, bark, and roots are reported to be antidotes for poisoning from Manihot esculenta (Crantz), Jatropha curcas L., and Hura crepitans L. . A decoction of the leaves is used to stop vomiting and nausea, as a mild diuretic, to treat heartburn, prostate, urinary difficulties, and stomach problems .
Scientific evidences of B. orellana bioactivity are nowadays numerous, particularly regarding seed and leaf extracts. Anticonvulsant, antidiabetic, cardioprotective, and antimicrobial activities of different B. orellana extracts have been proved [12–14]. Fleischer et al. demonstrated the antimicrobial activity of B. orellana leaf and seed extracts against a range of Gram-positive and Gram-negative bacteria and fungi . Moreover, some pharmacological studies have revealed that B. orellana extracts possess an antiprotozoal and anthelmintic effect . Due to their broad antioxidant and antibacterial activities, polar extracts from B. orellana leaves and seeds have been recently proposed as alternative natural preservatives in food matrices . Phytochemical investigations have revealed the presence of almost twenty-five types of chemical compounds from B. orellana different extracts, with the following major components: carbohydrates, carotenoids, steroids, proteins, flavonoids, terpenoids, phenolics, tannins, glycosides, alkaloids detected only in the leaf, and anthroquinones only in the seeds [15, 18].
Despite being widely used and studied, little is known about B. orellana volatile organic compounds (VOCs) composition, especially regarding the terpene profile from various plant organs. Moreover, even if B. orellana seed extracts claim to repel insects, little data for a clear validation is available. Therefore, the present research provides the first investigation on B. orellana chemical composition of different plant organs (fruits, leaves, bark, wood, and seeds) and the volatile profile sampled from living plants growing in an Amazonian Reserve (in vivo sampling) during an Italian-Brazilian cooperation project. In addition, a preliminary investigation on the repellent properties of seed extracts against A. aegypti L. was conducted.
2. Materials and Methods
2.1. Plant Samples
B. orellana plants growing in proximity of the Upper Rio Guamà Reserve located in the municipality of Capitao Poço, Parà State, Brazil (46°59′54.82′′ O; 1°52′4.08′′ S; 152 m. a.s.l.) were identified, and the volatile compounds emissions of fruits were in vivo sampled in February 2011 during a mission of an Italian-Brazilian cooperation project. Dry seeds, roots, leaves, bark, and wood were bought at Belem, at the Ver-o-Peso commercial district, from the most frequented herbalist shop (1°27′8.84′′S; 48°30′8.99′′O). Identification of the in vivo sampled plants and purchased materials was participatory and based on traditional knowledge of Indios Tembè, a tribe living in Itaputyr, a village in the upper Rio Guamà reserve [19, 20].
2.2. Standards and Chemicals
2,2-diphenyl-1-picrylhydrazyl (DPPH), the Folin-Ciocalteu reagent, sodium carbonate, methanol, and ethanol were purchased from Sigma-Aldrich, Milan, Italy.
Volatile compounds used as references were purchased from Sigma-Aldrich-Fluka, from the General and Flavours and Fragrances catalogue, Milan, Italy.
2.3. Superfine Grinding (SFG)
In order to obtain a representative plant sample a superfine powder was prepared from B. Orellana’s different organs (seeds, wood, bark, and leaves) using mechanical grinding activation in an energy intensive vibrational mill. Three g of dry plant material samples were ground in a high intensity planetary mill Retsch (model MM 400, Retsch, GmbH, Retsch-Allee, Haan), as previously reported . The mill was vibrating at a frequency of 25 Hz for 4 min using two 50 mL jars with 20 mm stainless steel balls. Precooling of jars was carried out with liquid nitrogen in order to prevent the temperature from increasing during the grinding process. The speed differences between balls and jar resulted in the interaction of frictional and impact forces, releasing high dynamic energies. The interplay of all these forces resulted in the very effective energy input of planetary ball mills.
2.4. Seed Extracts Preparation
Three different B. orellana seed extracts were prepared to evaluate antioxidant capacity and phenolic content.
2.5. Traditional Infusion
Aliquots of 1 g from B. orellana seed powder were infused for 15 min with 100 mL of freshly boiled distilled water and cooled to room temperature. The infusion was then filtered using Whatman No. 4 filter paper in order to remove plant residues, and the aliquots were stored at +4°C overnight until the analysis.
2.6. Exhaustive Extraction (H2O and EtOH)
Aliquots of 0.3 g of B. orellana seed powder were subjected, respectively, to sequential extraction using 8 mL of distilled water, sonicated for 10 minutes, macerated for 1 h, and then sonicated with an ultrasonic cleaning bath (Branson 2510, 20 KHz, 120 W) for a further 30 min at room temperature. The procedure was repeated eight times to obtain a final volume of 64 mL of extract. This similar procedure was repeated for new samples using 8 mL of EtOH solution. Extracts were then filtered using Whatman No. 4 filter paper, and the aliquots were stored at +4°C until further analysis.
2.7. 2,2Diphenyl-1-Picrylhydrazyl (DPPH) Assay
Antioxidant capacity of the extracts was measured by the stable radical DPPH as previously reported . Each extract was diluted with methanol to five different concentrations between 0.09 and 0.74 mg mL−1. The DPPH concentration in methanol was 0.1 g L−1. One mL of extract solution (sample) or methanol (control) was mixed with 1 mL of DPPH, and the absorption was recorded after 20 min in the dark, at 517 nm (UV/VIS spectrophotometer, model 7800, Jasco, Milan, Italy). Antioxidant capacity (AC) was calculated according to the following equation: where is the absorbance of the sample and is the absorbance of the control. The concentration of extracts causing a 50% decrease of DPPH solution absorbance (IC50) was calculated by linear regression of the concentration-response plots, where the abscissa represented the concentration (mg mL−1) of the extracts and the ordinate of the average percentage of AC from three separate determinations. The antioxidant capacity of all B. orellana extracts was expressed as 1/IC50 (mean value).
2.8. Determination of Total Phenol Content
The amount of total phenols in B. orellana extracts was determined using the Folin-Ciocalteu reagent according to the modified method of Slinkard and Singleton, 1977, using Gallic acid as a standard, as previously reported . Distilled water (1550 μL) was combined and vortexed with 50 μL of sample (4.6 mg dry w/mL) and 100 μL of Folin-Ciocalteu’s reagent. Then, 300 μL of Na2CO3 (20%) was added to reach a final concentration of 0.05 mg mL−1; the mixture was vortexed. The absorbance of all samples was measured at 765 nm using a UV/VIS spectrophotometer (model 7800, Jasco, Milan, Italy) after incubation at 40°C for 30 min. Quantification was done on the basis of the standard curve of Gallic acid (solution of Gallic acid 10% EtOH, 0.25–10 μg/mL). The calibration equation for Gallic acid was , . The results were expressed as milligrams of gallic acid equivalents (GAE) per gram of dry weight. Measurements were taken in triplicate and expressed as mean value.
2.9. Seed Extracts for Mosquito Repellency Activity
To evaluate mosquito repellency activity of B. orellana, different extracts from powder seeds were prepared using a 5-step extraction/drying procedure (30 mL solvent for each step) using three different solvents: ethanol, ethanol/water (50 : 50 v/v), and hexane, as reported in Table 1. Dry residues were stored at 4°C until resuspension in 5 mL of solvent for skin application (Table 2).
2.10. Repellent Activity Test
The method of percentage of protection in relation to the dose was used . Hundred blood-starved Aedes aegypti females (4–8 days old), reared at 25°C, 80% RH, and an L:D cycle of 12:12 hours, were kept in a net cage (45 × 30 × 45 cm). Before each test, the untreated forearm (control) of some experimenters was exposed to mosquitoes in the test chamber. Once the experimenters observed five mosquito landings on the untreated arm, they removed their arm from the chamber. The arms of the test person were cleaned with ethanol, washed with unscented neutral soap, thoroughly rinsed, and allowed to dry 10 min before extracts application. After air drying the arms of the tested person, only 25 cm2 ventral side of the skin on each arm was treated and exposed, while the remaining area was covered by rubber gloves and apposite covering bracelets. The different extracts of B. orellana seeds (at three different concentrations each) were applied until complete covertures of the skin in three different trials. The control and treated arms were introduced serially into the cage. The number of landings was counted over 3 min at 11:00 a.m, at 2:00 p.m and at 6:00 p.m to evaluate differences in mosquito appetite. The experiment was conducted by 3 human volunteer testers and replicated three times at each concentration. All experiments were carried out at a temperature of °C and RH % under laboratory conditions. No skin irritation from the tested seed extracts was observed.
The percentage of protection was calculated by using the formula:
2.11. Headspace Solid-Phase Microextraction (HS-SPME) Volatile Compounds Sampling from B. orellana Living Plants (In Vivo)
B. orellana opened fruits were sampled in vivo in order to evaluate the VOCs fingerprint emitted by living plants. Each fruit was enclosed in a customised Teflon cage (manufactured by SNK, Inc., Fullerton, CA: 11 × 21 cm) into which a manual SPME holder was inserted to extract the headspace. VOCs were collected using 50/30 μm Divinylbenzene/Carboxen/polydimethylsiloxane (CAR/PDMS/DVB) StableFlex fibre (Supelco; Bellefonte, PA). The fibre was exposed to the plant headspace for 8 h. Long sampling time were selected in order to obtain a comprehensive VOCs profile emitted by plants. Peaks originating from the cage (Teflon material) were also assessed by extracting a blank sample.
2.12. HS-SPME Volatile Compounds Sampling of Different Dried B. orellana Organs (Seeds, Wood, Bark, and Leaves)
The HS-SPME extraction conditions were optimised in our previous study on the characterisation of Achillea collina VOCs (selection of SPME fibre, sample amount, and extraction time) . Briefly, all samples were prepared by weighing exactly 1.00 g of B. orellana powdered samples of seeds, wood, bark, and leaves, respectively, in a 20 mL glass vial, fitted with cap equipped with silicon/PTFE septa (Supelco, Bellefonte, PA, USA), and by adding 1 mL of the internal standard solution (IS) in water (1,4-cineol, 1 μg/mL, CAS 470-67-7) to check the quality of the fibres.
At the end of the sample equilibration period (1 h), a conditioned (1.5 h at 280°C) 50/30 μm Divinylbenzene/Carboxen/polydimethylsiloxane(CAR/PDMS/DVB) StableFlex fibre (Supelco; Bellefonte, PA) was exposed to the headspace of the sample for extraction (3 h) by CombiPAL system injector auto-sampler (CTC analytics, Switzerland). Temperature of 30°C was selected as extraction temperature in order to prevent possible matrix alterations (oxidation of some compounds, particularly aldehydes). To keep a constant temperature during the analysis, the vials were maintained on a heater plate (CTC Analytics, Zwingen, Switzerland).
2.13. Gas Chromatography-Mass Spectrometry Analysis of VOCs
HS-SPME analysis was performed using a Trace GC Ultra (Thermo-Fisher Scientific; Waltham, MA, USA) Gas Chromatograph coupled to a quadruple Mass Spectrometer Trace DSQ (Thermo-Fisher Scientific; Waltham, MA, USA) and equipped with an Rtx-Wax column (30 m; 0.25 mm i.d.; 0.25 μm film thickness, Restek, USA). The oven temperature programme was from 35°C, hold 8 min, to 60°C at 4°C/min, then from 60°C to 160°C at 6°C/min, and finally from 160°C to 200°C at 20°C/min. Carry-over and peaks originating from the fibre were regularly assessed by running blank samples. After each analysis fibres were immediately thermally desorbed in the GC injector for 5 min at 250°C to prevent contamination. The injections were performed in splitless mode (5 min). The carrier gas, helium, at a constant flow of 1 mL/min. An n-Alkanes mixture (C8–C22, Sigma R 8769, Saint Louis, MO, USA) was run under the same chromatographic conditions as the samples to calculate the Kovats retention indices (KI) of the detected compounds . The transfer line to the mass spectrometer was maintained at 230°C, and the ion source temperature was set at 250°C. The mass spectra were obtained by using a mass selective detector with the electronic impact at 70 eV, a multiplier voltage of 1456 V, and by collecting the data at the rate of 1 scan/s over the m/z range of 30–350. Compounds were identified by comparing the retention times of the chromatographic peaks with those of authentic compounds analyzed under the same conditions when available or by comparing the Kovats retention indices with the literature data. The identification of MS fragmentation patterns was performed either by the comparison with those of pure compounds or using the National Institute of Standards and Technology (NIST) MS spectral database. Volatile compounds measurements from each headspace of the plant extracts were carried out by peak area normalization (expressed in percentages). All analyses were done in triplicate.
Data is expressed as mean value and standard deviation.
3. Results and Discussion
3.1. Antioxidant Capacity and Total Phenol Content Analysis
Results of the antioxidant capacity and total phenol content (TPC) of all B. orellana seed extracts (traditional infusion, H2O, and H2O/ETOH) are presented in Figure 1. The free radical scavenging activity was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method, which is regarded as an easy, reliable, sensitive, and rapid method. A review by Moon and Shibamoto also reported that almost 90% of antioxidant studies are conducted with this method . We noticed that the DPPH scavenging activity was significantly greater for the seeds extracted with water than for extracts obtained with infusion and ethanol. It is well known that antioxidants react with DPPH free radical and convert it to the stable form. Therefore, B. orellana seeds extracted with H2O seem to contain a great amount of antioxidant compounds which may act as primary antioxidants able to react with free radicals as efficient hydrogen donors. A broad antioxidant activity of leaf and seed polar extracts has been recently proved using five different test systems by Viuda-Martos et al. . Cardarelli et al. reported that the highest free radical scavenging capacity of annatto extracts was observed in the extract obtained with the most polar solvents, which also showed the highest phenol content .
In our conditions, the total phenol content resulted significantly higher for seed extracts obtained with H2O than for infusion or H2O/ETOH extraction.
This result clearly indicates that most of the phenols present in the plant are in a polar form. The TPC of B. orellana seed extracts, expressed as Gallic acid equivalent, ranged from 2.82 mg/g to 4.67 mg/g. Cardarelli et al. reported a lower TPC content that ranged between 0.30 and 1.84 mg of Gallic acid equivalent per gram of dry seeds (depending on the solvent used for extraction), while Chisté et al. recorded TPC values of 1.7 mg of Gallic acid equivalent per gram of wet seeds [26, 27]. The concentration and type of phenol substances present in the extracts are influenced by seasonal and environmental factors, for example, climate and soil type, genetic factors, and processing methods such as type of solvent used It is noteworthy that the water extractable phenol compounds present in B. orellana dry seeds are higher than that of green beans, tomatoes, green cabbage, green collard, and some green leafy vegetables. Bioactive phenols are very interesting as antioxidants because of their natural origin and their ability to act as efficient free radical scavengers, able to protect the cells against the oxidative damage caused by free radicals .
3.2. Repellent Activity
Blood-feeding insects are of great medical and veterinary importance, due both to their nuisance value and as vectors of disease . Amongst insects that feed on humans, mosquitoes are of global importance . In Amazonian regions, the use of plants as insect repellents is a common practice among Indio tribes. The majority of Itaputyr interviewed villagers admitted that they could not afford to use synthetic commercial mosquito repellents or insecticides because of high cost and/or discouraged by poor performance of some of the commercial products. Similar findings have been reported elsewhere in Africa, such as Guinea Bissau and Kenya, where the majority of the villagers could not afford synthetic commercial mosquito insecticides due to poverty [31, 32].
The results of mosquito protection in relation to application dose of different B. orellana dried seeds extracts are shown in Table 3. Skin repellent test showed the best results using a highly concentrated extract. Dried hexane extract resuspended in acetone provided the highest total percentage protection, followed by hydroalcoholic extract, with 90% and 73% of protection, respectively.
|Data expressed as mean ± standard deviation . |
These results support the presence of mosquito repellent compounds in B. orellana seeds, particularly in the nonpolar fraction, also suggested by the Indio tradition to use the seeds to paint their bodies during open air religious and war ceremonies.
3.3. Volatile Organic Compounds (VOCs) Analysis
The volatile compounds extracted from headspaces of different B. orellana organs are presented in Table 4. At present, this represents the first report on the characterization of volatile compounds from different B. orellana organs by using the SPME technique. The SPME technique has the advantage of minimizing the sample handling and consequently decreasing the loss of volatile compounds. It is a simple and fast modern tool used to characterize the VOCs fingerprint of aromatic and medicinal plants and offers a valid alternative to hydrodistillation . Moreover, this technique could represent a valid tool to analyze volatile compounds emitted by in vivo plants, sampling them in situ, in a nondisruptive way. Overall, 71 volatile compounds were identified in B. orellana samples with different proportions amongst the different plant organs.
|The Kovats retention index calculated for Rtx-Wax capillary column (25 m 0.25 m 0.25 mm i.d.).|
bNormalized amount of volatile compounds (percentage); (peak of volatile compound/total peak area of all volatile compounds) of B. orellana ) obtained from different parts.
cNormalized amount of volatile compounds (percentage); (peak of volatile compound/total peak area of all volatile compounds) of B. orellana living plants using in vivo sampling ().
s.d.: ± standard deviation.
nd: not detected.
The majority of volatile compounds present in the headspace from different B. orellana organs were sesquiterpenes, monoterpenes, hydrocarbons, and ketones. Simple terpenes have gained attention as agents of communication and defense against insects and can act as attractants or repellants.
Monoterpenes and sesquiterpenes are major components of many spices, fruit, and flower essential oils, having a great economic importance . In the present study, we found a very scarce presence of monoterpenes especially α-pinene, that was absent in the in vivo samples. This is in contrast with Galindo-Cuspinera et al. that found α-pinene to be a major component isolated in the oil obtained from B. orellana samples. However, the volatile compounds were recovered using dynamic headspace-solvent desorption sampling and analyzed using GC-MS .
Sesquiterpenes constituted the major group of volatile compounds found in all the different B. orellana extracts analyzed. The major constituents were D-germacrene, δ-elemene, γ-elemene followed by β-caryophyllene, α-caryophyllene, γ-muurolene, and δ-cadinene.
Sesquiterpenes were significantly higher than mono-di-terpenes in all plant organs analyzed. Leaves bark and seeds showed higher sesquiterpene concentration than wood and fruits dried or in vivo sampled. The total percentage of VOCs identified in the wood was rather low, only 61.59%, as compared to the other plant samples. Also, Zollo et al., found low levels of volatile compounds extracted from leaves and wood of B. orellana. The authors identified a great percentage of ishwarane, a sesquiterpene hydrocarbon not found in the present research .
Elemene and δ-cadinene have been found in all samples at different percentages. Minor sesquiterpenes found in B. orellana extracts that have distinctive aromas include cubebene, β-, and δ-cadinene. Cubebene has been described as having a fruity, sweet, citrus-like smell. β- and δ-cadinene are compounds occasionally used as fixatives in candy flavours and have a dry-woody, slightly medicinal-tarry odour with some similarity to spices in the cumin-thyme family . Some of the monoterpenes and sesquiterpenes found in our extracts have been previously described as having antimicrobial properties or as being associated with pharmacological properties . D-germacrene, mainly found in dried seeds and leaves samples (27.83% and 21.13%, resp.), showed antimicrobial activity .
β-caryophyllene, mainly found in in vivo samples (37.32%), has been proved to have an anti-inflammatory effect by selectively binding cannabinoid receptors type 2 (CB2) .
Cis-ocimene, mainly found in dried fruits samples (19.99%), showed antibacterial activity against Gram+ and Gram− (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae) .
This study has renewed the state of knowledge about B. orellana bringing together the existing literature information and novelties such as VOCs profiling and indications about mosquito repellent capacity of seed extracts. At present, this research represents the first investigation on volatile compounds conducted on different B. orellana organs by means of a fast sample preparation method. In addition, SPME was confirmed to be a good technique to analyze volatile compounds emitted by in vivo plants, sampled in situ, in a nondisruptive way, with consequent potential big advantages for phytochemical and ecophysiological studies, particularly regarding rare and/or protected plants. General screening confirmed the literature data about phenol compounds content and radical scavenging capacity of B. orellana seed extracts.
The preliminary results on mosquito repellent activity confirmed the potential of the plant to protect against A. aegypti representing another theme of future investigation, fundamental in linking traditional value of a well-known plant with scientifically obtained proof of efficacy.
Conflict of Interests
The authors declare that they have no conflict of interests.
The authors are grateful to the Itapury Cooperation Project for supporting the mission costs.
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